This application claims priority to application No. 94/09528, filed Aug. 1, 1994 in France and application No. 94/09529, filed Aug. 1, 1994 in France.
The present invention relates to a process for separating and/or detecting and/or quantifying (an) infectious compound(s) in a biological material and to a support for implementing the process.
According to the present invention, infectious compounds, generically referred to below in abbreviated form by xe2x80x9cICxe2x80x9d, are understood to mean both compounds, in particular proteinaceous compounds, which are constituents of an infectious agent and structures which include infectious compounds. These structures are, in particular, either complete or incomplete, endogenous or exogenous infectious agents, their metabolites or else assemblies which contain constituent compounds of these infectious agents, which assemblies exhibit certain properties of said infectious agents, in particular the property of being detected by certain antibodies which are specific for infectious compounds; the IC""s can also be compounds which are specifically induced in the organism by the previously defined IC""s or by the expression of genes which are being expressed in an abnormal manner. IC""s which may be mentioned, for example, are viruses, bacteria, fungi, mycoplasmas, parasites and abnormal animal cells. A viral infectious compound will be designated below by xe2x80x9cVICxe2x80x9d while a non-viral infectious compound, that is to say an infectious compound of a type other than solely viral, will be designated by xe2x80x9cnVICxe2x80x9d.
xe2x80x9cBiological materialxe2x80x9d is understood here to be a biological tissue, a liquid or solid preparation or extract derived from biological tissue, or a natural medium which is capable of containing an infectious compound in the above-defined sense (for example, drainage water). The material can also be a mixture of at least two materials as defined above. Such a biological material can, therefore, be, in particular, either prepared from tissues, organs, stools or biological liquids from a patient who is suffering from an infection, for example a viral, a bacterial, a parasitic, a mycotic or a mycoplasma infection, or obtained from xe2x80x9cin-vitroxe2x80x9d cultures; such a biological material can also be a serum, plasma, urine, cerebrospinal fluid, synovial fluid, peritoneal fluid, pleural fluid, seminal fluid or ascitic fluid.
It is known that xcex22-glycoprotein I, abbreviated xe2x80x9cxcex22GPIxe2x80x9d below, is a plasma glycoprotein whose sequence has been demonstrated, in particular, in the papers by J. LOZIER et al., Proc. Natl. Acad. Sci. ISA [sic], Vol. 81, pages 3640-3644, July 1984 and T. KRISTENSEN et al., FEBS Letters, Vol. 289, 1991, pages 183-186. xcex22GPI is also termed apolipoprotein H. It has been established that this protein exhibits a polymorphism: the name xcex22GPI will be regarded below as being generic for all the forms.
It has been shown in International Application WO 94/18569 that some viral compounds bind specifically to one form of xcex22GPI, namely that which is described in French Patent Application 2 701 263, whether this form of xcex22GPI is in a pure state or contained in a protein composition; this form of xcex22GPI is isolated from the residue which is bound to the affinity chromatography column(s) which is/are employed in the process for purifying albumin from blood plasma which is described in FR-A-2 690 444; it has a molecular weight of 50,000xc2x13000 daltons; in the context of the present patent application, this form of xcex22GPI has been designated in abbreviated form xe2x80x9cxcex22xe2x80x2GPIxe2x80x9d. A process has therefore been proposed in WO 94/18569 for detecting and/or assaying viral compounds, in which the viral compounds (VIC""s) are bound using xcex22xe2x80x2GPI. In such a process, the xcex22xe2x80x2GPI is therefore added to the VIC""s which are contained in a biological material in such a way as to separate the VIC""s which have thus been captured in order then to detect them and/or assay them.
In a general manner, a direct or indirect association between at least one IC and one xcex22GPI will be termed here xe2x80x9c(a) complex(es)xe2x80x9d: in a general manner, these complexes will be referred to below by the notation xe2x80x9cxcex22GPI/ICxe2x80x9d. The process described in WO 94/18569 detects and/or assays VIC/xcex22xe2x80x2GPI complexes between VIC viral compounds and xcex22xe2x80x2GPI which is either in a pure state or contained in a protein composition, with the xcex22xe2x80x2GPI being added to the biological material which contains the VIC""s to be detected and/or assayed.
The form or forms of xcex22GPI which is/are naturally present in the biological material prior to implementing the process described below, and not intentionally added as such, will be designated here (xcex22GPI)n. The form or forms of xcex22GPI which are added intentionally in order to form the previously defined IC/xcex22GPI complexes will be designated (xcex22xe2x80x2GPI)a.
According to the present invention, it was found, in a novel manner, that it was possible to separate, detect and/or quantify other IC/xcex22GPI complexes from a biological material than those described in WO 94/18569, namely:
(xcex22GPI)n/IC complexes where the IC part can be of the VIC viral type or the nVIC non-viral type and the (xcex22GPI)n part derives naturally from the biological material under study,
(xcex22GPI)a/nVIC complexes where the (xcex22GPI)a part is added intentionally and prepared in different pure or mixed forms for this purpose, and the nVIC part derives from non-viral infectious compounds in the biological material under study.
The present invention relates, therefore, to a process for separating and/or detecting and/or quantifying infectious compounds (IC""s) in a biological material, characterized in that a xcex22GPI/IC complex selected from the group formed by:
a) the (xcex22GPI)n/IC complexes
b) the (xcex22GPI)a/nVIC complexes
is separated and/or detected and/or quantified.
In a general manner, the xcex22GPI, like the IC""s, can, for some applications of the process, be of animal origin or produced by genetic and/or chemical engineering. The process can be applied both to man and to animals.
According to the invention, the xcex22GPI part of the complexes will be identified by its being recognized with the aid of (a) substance(s) which may bind preferentially, or which binds specifically, to this part, and the IC part will be identified by any suitable means.
The formation of the complexes can be direct or indirect and can be mediated or promoted by certain additives, which can be chemical, biochemical or biological, such as certain lipids or detergents, in particular phospholipids. The complexes can be formed during the preparation of the biological material and/or during one (of the) stages of the process.
As previously pointed out, the IC""s comprise the VIC""s and the nVIC""s. The VIC""s which may in particular be mentioned are those of the group formed by HIV1, HIV2, HBV, HSV and particles or proteins of viral origin; those nVIC""s which may in particular be mentioned are those of the group formed by bacteria, parasites, fungi and mycoplasmas and, more specifically: in the case of bacteria: Borellia; and in the case of parasites: Leishmania, infantum in particular, Toxoplasma gondii and Entamoeba histolytica.
Advantageously, the xcex22GPI which is retained in the complex(es) may or may not, according to the embodiments of the process, have been labeled; this labeling, which may or may not take place beforehand, can be effected, for example, by means of an antibody, an enzyme, a radioactive product, a fluorescent product or a metallic agent.
According to the present invention, a poly-specific test which is able to detect different infectious agents can be carried out using, in particular, simultaneously or successively, a different detection method for each agent, for example an alkaline phosphatase-conjugated antibody against HIV2p26 and then a peroxidase-conjugated antibody against HBsAg.
In a first subset of embodiments of the present invention, in which an external (xcex22GPI)a is added to the biological medium, use can be made of xcex22xe2x80x2GPI, which is pure or is in the form of a protein composition which contains, in particular, other glycoproteins, as the xcex22GPI form for the xcex22GPI part of the complex; this composition can, in particular, be that which is obtained by eluting a gel affinity column which carries sulfate groups, as described in French Patent Application 2 701 263. However, other forms of xcex22GPI may also be employed, for example that obtained in accordance with the protocol described by J. Arvieux et al. in Journal of Immunological Methods (1991), 143, pp. 223-229. Carbamylated xcex22GPI is able to form some complexes.
In the embodiments which belong to the previously defined first subset, complexes are formed by adding the (xcex22GPI)a to a biological material in order to separate and/or assay and/or quantify nVIC""s.
According to a second subset of embodiments of the process according to the invention, use is made of the (xcex22GPI)n which is naturally and/or initially present in a biological material. It is known that the process of WO 94/18569 yields satisfactory results when the biological material contains free VIC""s, that is VIC""s which are not attached to the xcex22GPI which is naturally present in the biological material, with said free VIC""s consequently being able to attach themselves to the xcex22xe2x80x2GPI which is added in accordance with this process. In this process, the response signal therefore increases as the VIC""s increase which possess sites which are still free or which may have been released by competition from complexes which are naturally present in the biological material. On the other hand, it is possible to assume, in some cases, for example at the beginning of an infection, that the VIC""s are present in a quantity which is low as compared with the (xcex22GPI)n which is naturally present and that said VIC""s are therefore in the main bound to the (xcex22GPI)n in the form of (xcex22GPI)n/IC complexes. The test proposed in Application WO 94/18569 may not in that case be meaningful since the VIC""s which have thus been complexed may be masked. According to this second subset of the invention, it is proposed, therefore, to observe and/or separate and/or detect and/or quantify IC""s which are present in a biological material, in particular in the case where these compounds are in a quantity such that, as compared with the (xcex22GPI)n which is normally present, they are in the main complexed (or complexable) with at least one of the forms of (xcex22GPI)n, or else in the case where said IC""s cannot be displaced by introducing (xcex22GPI)a.
Given the fact that, according to this second subset of embodiments according to the invention, no xcex22GPI has been added, the masking phenomenon of the process of WO 94/18569 is avoided and a response signal is obtained which can increase as a function of the quantities of complex, and therefore of IC, which are contained in the biological material, even in the case of very low quantities of these IC""s. Where appropriate, the process can make it possible to detect an initial state of pathology, whereas the earlier process is more appropriate for studying an established pathological state, corresponding, for example, to an excess of IC or to a deficiency of (xcex22GPI)n or to the natural existence of a form of xcex22GPI which is non-functional as regards binding to the IC(s).
It is to be noted that these two embodiment subsets are not mutually exclusive.
In order to implement the process according to the invention, IC""s can be detected without prior attachment of the xcex22GPI/IC complex to a support or with attachment of said complex to a support by means of an element which is contained in the complex; in the first case, the detection and/or the quantification is effected in the medium in which the complex is formed, either after the said medium has been attached by means of a physical, chemical or biochemical method, for example to a surface, or without attachment of said medium; in the second case, the support can, advantageously, be a solid support.
In the present description, the term attachment to the support is used without prejudging the time at which the complex is formed.
According to one variant of the invention, the xcex22GPI/IC complex is held by means of the xcex22GPI part of the complex, by providing a support with a compound which binds to the said xcex22GPI part; the part of the complex corresponding to the IC""s is then separated/detected/quantified by an appropriate means.
According to another variant of the invention, the xcex22GPI/IC complex is held by means of the IC part of said complex, by attaching the latter to a support which is provided with a compound which binds to said IC part of the complex; the xcex22GPI part of the said complex is then detected and/or separated and/or quantified by any appropriate means, advantageously with the aid of (an) antibody(ies) which is/are, in particular, conjugated and is/are specific for xcex22GPI. The presence in the native state, or the addition, of (a) detergent(s) or lipid(s) can help to attach these antibodies.
According to the invention, the detection can be effected by visualizing and/or counting a structure which is characteristic for the IC, in particular with the eye or by means of microscopy (in particular optical, electron or UV microscopy), by means of detecting the (xcex22GPI)n which is associated with the IC(""s) on these structures, in particular with the aid of a specific labeled antibody, for example an antibody which is conjugated to an enzyme molecule or a fluorescent molecule. The IC(""s), which may or may not be complexed, of the biological material can be physically, chemically or biologically attached to a support or be in liquid (in particular acid, ketonic, alcoholic, paraffinic or aldehydic liquid) phase. A double label which is specific for each part can also be employed for detecting the complex, in particular using antibodies which are specific for each part and which are conjugated to 2 different tracers, for example tracers-which fluoresce at different wavelengths. Finally, the IC""s in the biological material can be detected or assayed with the aid of an apparatus for analyzing signals such as number, volume, size or shape of particles or structures, for example by means of flow cytometry, in particular.
According to another variant of the invention, the xcex22GPI/IC complex is held on a support by means of a compound which binds to either the xcex22GPI part or the IC part of the complex, after which the complex which is attached to said support is separated from said biological material and said (IC/xcex22GPI) complex is separated and/or detected and/or quantified and/or assayed by recognition of the other part of the complex.
The support is advantageously a solid support: this can be a membrane, for example a nitrocellulose membrane, or a microtitration plate, for example an ELISA microtitration plate, or a microscope slide.
The compound which binds to one of the parts of the complex, an antibody for example, is attached to the support by reacting reactive groups of said compound with the reactive sites of the support. Preferably, this reaction is effected at a temperature of between 0 and 40xc2x0 C.; the compound which binds to one of the parts of the complex is, advantageously, placed in a buffer which has a pH of between 4.5 and 10.5, preferably of between 6.5 and 7.5; this buffer can advantageously be of the phosphate or acetate type. The support is advantageously maintained in contact with the buffer which contains the compound at a temperature of between 0 and 40xc2x0 C. for an incubation time of between 30 min and 24 hours. After incubation, the buffer, which has not reacted, is separated from the support and the support is washed, preferably with a buffer which is identical to the previous buffer apart from the fact that it does not contain the abovementioned compound. It may be necessary to saturate the active sites of the support which have not reacted with said compound; in this case, other active groups are caused to react with these active sites; for this purpose, use is advantageously made of a solution of serum albumin, for example bovine serum albumin, or of casein and/or of polyvinylpyrrolidone and/or of gelatin and/or of detergent, which are used simultaneously or successively. After reaction, the support is preferably rinsed and dried.
The support to which the compound which binds to the complex is attached is then placed in contact with a biological material, in particular a liquid, containing the sought-after IC. This biological material is advantageously diluted with the aid of a buffer which gives a pH of between 4.5 and 10.5, preferably of between 5.6 and 7.5. The reaction on the support is preferably effected at a temperature of between 0 and 40xc2x0 C., advantageously in the vicinity of 37xc2x0 C., for a period whose duration is between 30 minutes and 24 hours. The solution containing the IC which has not reacted is then advantageously separated from the support; where appropriate, the support is then washed with a saline solution, preferably a buffered saline solution.
In the case where the attachment of the complex to a support is effected by means of the xcex22 GP I part of the complex, the compound which binds to xcex22 GP I can be an antibody which recognizes the xcex22 GP I, or be another protein, for example of viral origin or prokaryotic or eukaryotic cellular origin, such as an albumin or a biological compound, for example a fatty acid, or a lipid, such as a phospholipid, or a chemical compound, for example dextran sulfate, heparin sulfate or a detergent. A free radical or activated radical of the support can bind the xcex22GPI, sometimes preferentially.
The IC of the xcex22GPI/IC complex which is bound to the support by the xcex22GPI can be separated and/or assayed and/or quantified by any known means, such as infectivity, a specific enzyme reaction, a tracer, for example a fluorescent or radioactively labeled tracer, detection of specific nucleic acids by hybridization with a labeled probe, a polymerase chain reaction (xe2x80x9cPCRxe2x80x9d), an assay, counting, visualization or an optical or (electron) microscopic method. However, the detection and/or assay is/are preferably effected with the aid of an antibody which specifically recognizes proteins of the IC""s to be detected. In a known manner, this antibody can be conjugated to an enzymic label, for example peroxidase; the excess of antibody is eliminated by washing; a substrate which is specific for the enzyme which is conjugated to the antibody is then added, in a known manner, with the substrate being transformed, under predetermined conditions, into a colored product; the formation of said colored product indicates the presence of the sought-after IC and enables this IC to be assayed. Use can also be made of an antibody against the IC which is coupled to an isotopic label and then detected radio-metrically.
In the case where the attachment to the support is effected by way of the IC part of the complex, specifically if specific detection is desired, it is visualized with xcex22GPI which is advantageously conjugated to a label. The IC can be attached to the support either directly or indirectly, for example by way of an antibody. Advantageously, the label can be an enzyme, a radioactive product or a fluorescent product. The IC can be attached to the support by reacting reactive groups of the IC with the reactive sites of the support when the attachment is direct, or by attaching a compound, for example an antibody, to the reactive sites of the support and attaching the IC to said compound which has previously been attached to the support.
While the xcex22 GP I part of the complex, which complex has been attached by way of a compound which binds the IC part, can be detected by any appropriate means, it is advantageously detected using antibodies which are specific for the xcex22 GP I and which are, for example, conjugated.
The invention also relates to a solid support for implementing the above-defined process, characterized in that it is suitable for attaching one of the elements of the xcex22GPI/IC complex or a substance which is suitable for attaching one of the elements of said complex.